Thrombin-induced endothelial cell surface ICAM-1 expression plays a critical role in the pathogenesis of lung vascular injury. Signaling via the transcription factor NF-kappaB is required for the stable thrombin-induced ICAM-1 expression in endothelial cells. In Project 3, we will address the role of thrombin-induced Ca2" entry in the mechanism of NF-KB activation and signaling ICAM-1 expression and neutrophil (PMN) adhesion-mediated lung vascular injury. Our studies indicate that the thrombin-induced increase in intracellular Ca2+ concentration is dependent on both Ca2+ store depletion and the Ca2+ store depletion-mediated Ca2+ influx. Further, we have shown that thrombin-induced Ca2+ influx occurs via transient receptor potential channels (TRPCs) expressed in endothelial cells. TRPC4 gene deletion (TRPC4-/-) in mice prevented thrombin-induced Ca2+ influx. In Supporting Data, we show using pharmacological and gene knockout approaches that the thrombin-induced Ca2+ influx is critically involved in signaling NF-kappaB activation and ICAM-1 expression in lung endothelial cells. Moreover, inhibition of Ca2+-dependent PKC-alpha markedly reduced the thrombin-induced ICAM-1 expression. However, the inhibition of calcineurin (Ca2+/ calmodulin-dependent phosphatase) increased thrombin-induced ICAM-1 expression in pulmonary endothelial cells. Based on these preliminary results, in Aim 1, we will test the hypothesis that TRPC-mediated Ca2+ influx regulates NF-kappaB activation, ICAM-1 expression, and PMN adhesion to pulmonary vascular endothelial cells. In Aim 2, we will test the hypothesis that the Ca2+ activation of PKC-alpha is required for ICAM-1 gene transcription via NF-kappaB activation. In Aim 3, we will test the hypothesis that thrombin-induced calcineurin activation acts in a negative feedback manner to inhibit ICAM-1 gene expression by preventing NF-kappaB activation. In Aim 4 studies, we will test the hypothesis that NF-kappaB activated TRPC1 expression augments Ca2+ influx, and the resultant ICAM-1 expression and PMN adhesion to pulmonary vascular endothelial cells. Studies will be carried out using endothelial cell culture, genetically modified mice (e.g., TRPC4-/- mice), and intact lungs. With these studies, we will be able to provide novel insights into the role of Ca2+ influx in endothelial cells in the mechanism of lung vascular injury and tissue inflammation.